高内涵细胞成像系统在细胞增殖检测中的应用
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摘要
目的将高内涵细胞成像增殖检测与胸腺嘧啶核苷(~3H-TdR)参入法、四甲基偶氮唑盐(MTT)法和细胞计数试剂盒-8(CCK-8)法等经典的细胞增殖检测方法进行比较。方法以成纤维样滑膜细胞(FLS)为研究对象,采用常用的增殖刺激剂肿瘤坏死因子-α(TNF-α)和已知的增殖抑制剂甲氨蝶呤不同浓度处理细胞,观察不同方法检测FLS增殖的敏感性和稳定性。结果~3H-TdR参入法和高内涵细胞成像法可检测到1 nmol·L~(-1)MTX对FLS细胞增殖的明显抑制作用,MTT法和CCK-8法可检测出10 nmol·L~(-1)MTX对FLS细胞增殖的明显抑制作用。~3H-TdR参入法检测得到的数据组内离散程度较大,标准差为均值的32.61%~61.36%,MTT法为11.9%~17.8%,CCK-8法为17.15%~32.88%,高内涵细胞成像法为12.66%~26.54%。结论高内涵细胞成像为一种更准确和稳定的检测细胞增殖的实验方法。
Aim To compare high-content cell imaging system and other methods in detecting cell proliferation,including the traditional thymidine(~3H-TdR) incorporation method,methyl thiazolyl tetrazolium( MTT) method and cell counting kit-8( CCK-8) method.Methods The fibroblast-like synovial cells( FLS) were used as the study object to observe the sensitivity and stability of FLS proliferation in different methods by using the usual proliferative stimulant tumor necrosis factor-α( TNF-α) and the known proliferation inhibitor methotrexate at different concentrations.Results The ~3H-TdR method and high-content cell imaging could detect a significant inhibitory effect of 1 nmol·L~(-1) MTX on FLS cell proliferation,MTT assay and CCK-8 method could detect the significant inhibitory effect of 10 nmol·L~(-1) MTX on FLS cell proliferation.~3H-TdR method was found to have a large degree of discretization in the data set,with a standard deviation of 32.61%~61.36%,and the MTT method was 11.9%~17.8%, the CCK-8 method was 17.15%~32.88%,and the high-content cell imaging system method was 12.66%~26.54%.Conclusion The method of high-content cell imaging system is more accurate and stable for detecting cell proliferation.
Aim To compare high-content cell imaging system and other methods in detecting cell proliferation,including the traditional thymidine(~3H-TdR) incorporation method,methyl thiazolyl tetrazolium( MTT) method and cell counting kit-8( CCK-8) method.Methods The fibroblast-like synovial cells( FLS) were used as the study object to observe the sensitivity and stability of FLS proliferation in different methods by using the usual proliferative stimulant tumor necrosis factor-α( TNF-α) and the known proliferation inhibitor methotrexate at different concentrations.Results The ~3H-TdR method and high-content cell imaging could detect a significant inhibitory effect of 1 nmol·L~(-1) MTX on FLS cell proliferation,MTT assay and CCK-8 method could detect the significant inhibitory effect of 10 nmol·L~(-1) MTX on FLS cell proliferation.~3H-TdR method was found to have a large degree of discretization in the data set,with a standard deviation of 32.61%~61.36%,and the MTT method was 11.9%~17.8%, the CCK-8 method was 17.15%~32.88%,and the high-content cell imaging system method was 12.66%~26.54%.Conclusion The method of high-content cell imaging system is more accurate and stable for detecting cell proliferation.
引文
[1]杨思民,汪庆童,刘亢亢,等.重组TACI-Ig融合蛋白抑制抗CD3抗体诱导T淋巴细胞增殖与活化[J].安徽医科大学学报,2016,51(7):919-25.[1]Yang S M,Wang Q T,Liu K K,et al.Inhibitory effect of rh TACIIg on anti-CD3 antibody induced T lymphocytes proliferation and activation[J].Acta Univ Med Anhui,2016,51(7):919-25.
[2]Wang Y J,Zhou S M,Xu G,Gao Y Q.Interference of phenylethanoid glycosides from Cistanche tubulosa with the MTT assay[J].Molecules,2015,20(5):8060-71.
[3]Wang Q,Wang L,Wu L,et al.Paroxetine alleviates T lymphocyte activation and infiltration to joints of collagen-induced arthritis[J].Sci Rep,2017,7:45364.
[4]Gelles J D,Edward Chipuk J.Robust high-throughput kinetic analysis of apoptosis with real-time high-content live-cell imaging[J].Cell Death Dis,2016,7(12):e2493.
[5]Wu H,Chen J,Wang Q,et al.Ginsenoside metabolite compound K attenuates inflammatory responses of adjuvant-induced arthritis rats[J].Immunopharmacol Immunotoxicol,2014,36(2):124-9.
[6]Straume T,Raabe O G,Walsh K J,Wiley L M.Transmitted proliferation disadvantage from mouse oocytes labeled in vivo with[3H]thymidine:radiosensitive target considerations[J].Mutat Res,1997,374(1):11-9.
[7]岳晓莉,李萍,六欣,等.黄芪多糖对过氧化氢刺激皮肤成纤维细胞中线粒体和溶酶体的保护作用[J].中国病理生理杂志,2008,24(4):777-82.[7]Yue X L,Li P,Liu X,et al.Protective effect of astragalus polysaccharide on mitochondria and lysosome in H2O2stressed skin fibroblasts[J].Chin J Pathophysiol,2008,24(4):777-82.
[8]Jaszczyszyn A,Gasiorowski K.Limitations of the MTT assay in cell viability testing[J].Adv Clin Exp Med,2008,17(5):525-9.
[9]佟丽,辛增辉,陈育尧,等.Mtb诱导的SD大鼠佐剂性关节炎模型的建立及评价[J].中国药理学通报,2009,25(2):259-63.[9]Tong L,Xin Z H,Chen Y Y,et al.Establishment and evalution of adjuvant induced arthritis animal model by Mtb in Sprague-Dawley rats[J].Chin Pharmacol Bull,2009,25(2):259-63.
[10]Sieprath T,Corne T,Robijns J,et al.Cellular redox profiling using high-content microscopy[J].J Vis Exp,2017,(123).doi:10.3791/55449.
[2]Wang Y J,Zhou S M,Xu G,Gao Y Q.Interference of phenylethanoid glycosides from Cistanche tubulosa with the MTT assay[J].Molecules,2015,20(5):8060-71.
[3]Wang Q,Wang L,Wu L,et al.Paroxetine alleviates T lymphocyte activation and infiltration to joints of collagen-induced arthritis[J].Sci Rep,2017,7:45364.
[4]Gelles J D,Edward Chipuk J.Robust high-throughput kinetic analysis of apoptosis with real-time high-content live-cell imaging[J].Cell Death Dis,2016,7(12):e2493.
[5]Wu H,Chen J,Wang Q,et al.Ginsenoside metabolite compound K attenuates inflammatory responses of adjuvant-induced arthritis rats[J].Immunopharmacol Immunotoxicol,2014,36(2):124-9.
[6]Straume T,Raabe O G,Walsh K J,Wiley L M.Transmitted proliferation disadvantage from mouse oocytes labeled in vivo with[3H]thymidine:radiosensitive target considerations[J].Mutat Res,1997,374(1):11-9.
[7]岳晓莉,李萍,六欣,等.黄芪多糖对过氧化氢刺激皮肤成纤维细胞中线粒体和溶酶体的保护作用[J].中国病理生理杂志,2008,24(4):777-82.[7]Yue X L,Li P,Liu X,et al.Protective effect of astragalus polysaccharide on mitochondria and lysosome in H2O2stressed skin fibroblasts[J].Chin J Pathophysiol,2008,24(4):777-82.
[8]Jaszczyszyn A,Gasiorowski K.Limitations of the MTT assay in cell viability testing[J].Adv Clin Exp Med,2008,17(5):525-9.
[9]佟丽,辛增辉,陈育尧,等.Mtb诱导的SD大鼠佐剂性关节炎模型的建立及评价[J].中国药理学通报,2009,25(2):259-63.[9]Tong L,Xin Z H,Chen Y Y,et al.Establishment and evalution of adjuvant induced arthritis animal model by Mtb in Sprague-Dawley rats[J].Chin Pharmacol Bull,2009,25(2):259-63.
[10]Sieprath T,Corne T,Robijns J,et al.Cellular redox profiling using high-content microscopy[J].J Vis Exp,2017,(123).doi:10.3791/55449.